Somatotropin or growth hormone (“GH”) is a mammalian protein comprising a class of tropic hormones synthesized and secreted in the brain by the major gland of the endocrine system, the adenohypophysis. The secretion of GH and other tropic hormones by the adenohypophysis regulates the activity of cells in other endocrine glands and tissues throughout the body. Specifically, GH is secreted by somatotrophs of the anterior pituitary gland and functions to stimulate the liver and other tissues to synthesize and secrete IGF-1, a protein that controls cell division, regulates metabolic processes and exists in a free state or binds to one of six other proteins designated as IGFBP-1 through 6. The secretion process itself is modulated by opposing actions of somatoliberin (promoting GH release) and somatostatin (inhibiting GH release).
Human growth hormone (“hGH”) is of particular interest because it serves as a critical hormone in the regulation of cell and organ growth and in physiological function upon various stages of aging. For example, overproduction of hGH results in gigantism in children and acromegaly in adults, whereas under-production leads to dwarfism in children [Mauras et al., J. Clin. Endocrinology and Metabolism, 85(10), 3653-3660 (2000); Frindik et al., Hormone Research, 51(1), 15-19 (1999); Leger et al., J. Clin. Endocrinology and Metabolism, 83(10), 3512-3516 (1998)], Turner's Syndrome (females only) [Bramswig, Endocrine, 15(1), 5-13 (2001); Pasquino et al., Hormone Research, 46(6), 269-272 (1996)] and chronic renal insufficiency [Carroll et al., Trends in Endocrinology and Metabolism, 11(6), 231-238 (2000); Ueland et al., J. Clin. Endocrinology and Metabolism, 87(6), 2760-2763 (2002); Simpson et al., Growth Hormone & IGF Research, 12, 1-33 (2002)]. In adults, hGH deficiency can affect metabolic processing of proteins, carbohydrates, lipids, minerals and connective tissue and can result in muscle, bone or skin atrophy [Mehls and Haas, Growth Hormone & IGF Research, Supplement B, S31-S37 (2000); Fine et al., J. Pediatrics, 136(3), 376-382 (2000); Motoyama et al., Clin. Exp. Nephrology, 2(2), 162-165 (1998)]. Other hGH deficiency disorders characterized by growth failure include AIDS wasting syndrome [Hirschfeld, Hormone Research, 46, 215-221 (1996); Tritos et al., Am. J. Medicine, 105(1), 44-57 (1998); Mulligan et al., J. Parenteral and Enteral Nutrition, 23(6), S202-S209 (1999); Torres and Cadman, BioDrugs, 14(2), 83-91 (2000)] and Prader-Willi syndrome [Ritzen, Hormone Research, 56(5-6), 208 (2002); Eiholzer et al., Eur. J. Pediatrics, 157(5), 368-377 (1998)].
To date, treatment regimens for hGH deficiency in humans focus primarily on subcutaneous injection of purified hGH made by recombinant DNA technology. That therapeutic is packaged as either a solution in a cartridge or a lyophilized powder requiring reconstitution at the time of use. The frequency of injection varies depending on the disease being treated and the commercially available product being used. For example, dwarfism is treated by daily subcutaneous injection of recombinant hGH.
The use of subcutaneous administration as a rapid delivery route for hGH is necessitated by the inherent instability of the protein in solution. That instability results from cleavage of critical intramolecular crosslinks at specific positions within the amino acid sequence of the protein, which in turn disrupts the essential three-dimensional structure recognized by and associated with cellular surfaces in the patient. The mechanism for hGH cleavage or degradation is orchestrated primarily by oxidation of methionine residues or deamidation of aspartic acid residues upon dissolution, thereby rendering the protein inactive. Due to this fragility, a need in the art exists for hGH compositions or formulations that are stable and long-acting and can be delivered not only subcutaneously but by other conventional dosage routes, such as oral, dermal and intravenous routes.
A number of commercially available hGH products have been developed in an attempt to address this need. For example, NUTROPIN DEPOT® human growth hormone is an injectable suspension of recombinant human growth hormone (rhGH) embedded in a polylactide-coglycolide (PLG) microspheres (see www.gene.com). In addition to rhGH and PLG, the microspheres also comprise zinc acetate and zinc carbonate components. Prior to administration, the solid material must be reconstituted with an aqueous solution comprising carboxymethylcellulose sodium salt, polysorbate, sodium chloride and water. This suspension, which is mostly comprised of polymer, is administered once or twice monthly and requires a 21 gauge needle for injection. Due to the size of the microspheres and the viscous nature of the product, adverse injection-site reactions can occur, resulting in nodules, erythema, pain, bruising, itching, lipoatrophy and puffiness (see www.genentech.com/gene/products/inforrnation/opportunistic/nutropin-depot/index sp).
Another hGH product in development but subsequently discontinued is ALBUTROPIN™ human growth hormone, a long acting genetically produced fusion protein of human albumin and human growth hormone (see www.hgsi.com/products/albutropin.html). This product is said to exhibit prolonged half-life in circulation, roughly a fifty percent increase over that of soluble native hGH. ALBUTROPIN™ human growth hormone is typically delivered by injection on a weekly basis and is said to stimulate IGF-1 levels long after clearance from the body. The biological effect of this product is similar to that of currently available growth hormone therapies.
Another product developed was INFITROPIN CR™ human growth hormone, a formulation of hGH comprised of polyethylene glycol-conjugated hGH molecules. This conjugated hGH required a once a week injection and was said to be released at a continuous rate, without significant burst effect [Ross et al., J. Biol. Chem., 271(36), 21696-21977 (1996)]. However, this product was discontinued.
U.S. Pat. Nos. 5,981,485 and 6,448,225 refer to aqueous formulations of hGH that are said not to require a reconstitution step and are administered by daily injection. Such formulations typically contain hGH, a buffer, a non-ionic surfactant and optionally, a neutral salt, mannitol, or a preservative.
Various other drug delivery technologies, such as hydrogels [Katakam et al., J. Controlled Release, 49(1), 21-26 (1997)], liposomes, oil emulsions and biodegradable polymer microspheres, have been used in attempts to provide sustained drug release of hGH. However, the resulting formulations display a burst release of the drug, use harsh conditions and some are complicated to manufacture. This is especially true of hGH formulations based on DL-lactic co-glycolic acid (PLGA) microsphere technology, because the process used to produce the microspheres tends to employ conditions such as elevated temperatures, surfactant, organic solvents and aqueous/organic solvent interface, all of which cause protein denaturation [Herberger et al., Proc. Intl. Symp. Controlled Release of Bioactive Materials, 23, 835-836 (1996); Kim et al., Intl. J. Pharmaceutics, 229(1-2), 107-116 (2001)].
Some of the above-described preparations require hGH to be stored in a lyophilized state, which can be a time consuming and expensive process. U.S. Pat. Nos. 5,780,599 and 6,117,984 refer to divalent cation crystals of hGH and methods of producing divalent cation crystals of hGH, without the need for a lyophilization step.
Despite the efforts to address drawbacks of conventional hGH products, including instability upon storage and injection, short in vivo half-life, burst effects, lack of oral bioavailability, and difficulty and frequency of administration, the need for improved hGH preparations remains. To address this need, the present invention advantageously provides crystals of human growth hormone that yield stable, long-acting hGH.